High expression 5'utr-hhp1 sequence and application thereof

Abstract

The application provides a high-expression 5'UTR-HHP1 sequence and application thereof, and belongs to the technical field of medicines.The DNA sequence is shown as SEQ ID NO.1.The RNA sequence is shown as SEQ ID NO.2.The 5'UTR of a gene with extremely high expression efficiency in human cells and the natural 5'UTR sequence of a pathogen mRNA are found through a NCBI database.The ribosome load of candidate UTRs is predicted and optimized, and secondary structure prediction is performed through a Baidu PaddleHelix platform, and the UTRs with better prediction effects are selected for verification.A DNA-RNA two-dimensional verification system is established.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a highly expressed 5'UTR-HHP1 sequence and its applications. Background Technology

[0002] In recent years, mRNA technology has made groundbreaking progress. Gene expression structures consist of multiple elements: promoters (which may include enhancers), 5′ untranslated regions (5′UTR), protein-coding regions, 3′UTR, and PolyA signaling. However, research on the optimization of untranslated regions (UTR) remains relatively insufficient. UTR plays a crucial role in regulating mRNA translation efficiency and stability, especially the 5′UTR, which directly affects ribosome loading and translation initiation.

[0003] Currently, most commonly used 5'UTRs are derived from the hemoglobin gene, but studies have shown that designing new UTR sequences can further enhance protein expression levels.

[0004] In the 1980s and 90s, Kozak's team systematically revealed the regulatory mechanism of mRNA 5'UTR secondary structure on translation efficiency through a series of studies. Early experiments in the 1980s found that in Cos7 cells, when a moderately stable hairpin structure with a ΔG value of -30 kcal / mol existed in the 5'UTR, translation efficiency was not significantly affected; however, when the stability of the hairpin structure increased to -50 kcal / mol, regardless of its distance from the cap structure or the start codon, translation efficiency plummeted by 85%-95%. This phenomenon suggests that there is a definite energy threshold for ribosome tolerance to RNA secondary structures.

[0005] Subsequent studies, using a rabbit reticulocyte in vitro translation system, further elucidated the mechanism by which the spatial distribution of secondary structures influences translation. Experiments in 1989 confirmed that when a hairpin structure with ΔG = -30 kcal / mol was located 12 nt downstream of the cap structure, it significantly inhibited the initial binding of the 40S ribosomal subunit; however, when a structure of similar stability moved down to 52 nt from the cap, it had no effect on the translation process. This indicates that the structural accessibility of the ribosome binding site plays a decisive role in translation initiation, and once the 40S subunit successfully anchors to mRNA, it possesses the ability to unwind downstream secondary structures. In 1988, the team also discovered a key regulatory strategy: inserting an unstructured sequence upstream of the secondary structure element in the 5'UTR significantly improved translation efficiency, and this effect was positively correlated with the length of the inserted fragment. This provided an important theoretical basis for artificially optimizing mRNA translation efficiency.

[0006] In fact, the 5'UTR sequence has two main functions: enhancing mRNA stability and initiating translation. There are four strategies for optimizing the 5'UTR sequence in the development of mRNA vaccines. The first, and simplest, is to directly select the 5'UTR of genes with extremely high expression efficiency in human cells, such as the 5'UTR of human α-globin genes. The second is the natural 5'UTR of the pathogen's mRNA itself. Both methods are based on the inference that 5'UTRs optimized through natural selection will definitely function in muscle cells. The third is to achieve the phylogenetic evolution of 5'UTRs through logarithmic enrichment; this method was actually first used for screening 3'UTRs. The fourth is to perform high-throughput screening from massive databases using deep learning models. However, in terms of the speed of vaccine development, the first two methods are the fastest; the latter two require a significant amount of time and have certain technical difficulties.

[0007] BNT162b2 directly selects the 5'UTR of human α-globin mRNA and replaces the original background sequence (ACCATG) of the start codon ATG with the Kozak conserved sequence (GCCACCATG). Selecting the 5'UTR of a gene with extremely high expression efficiency in human cells also has the advantage of eliminating the presence of upstream AUGs that interfere with translation initiation in the 5'UTR region.

[0008] The secondary structure of native UTRs may inhibit translation (e.g., stem-loop structures adjacent to the cap structure) and exhibits instability across different genes or cell types. Furthermore, native sequences lack universality, necessitating re-screening for different target genes. Additionally, the expression mechanisms of plasmids and mRNA differ significantly (e.g., nuclear transcription versus direct cytoplasmic translation), resulting in low correlation between screening results and actual mRNA expression efficiency. Existing models rely on limited structural and expression data, leading to insufficient predictive accuracy and requiring extensive experimental validation. Summary of the Invention

[0009] The purpose of this invention is to propose a highly expressed 5'UTR-HHP1 sequence and its application. The 5'UTRs of genes with extremely high expression efficiency in human cells and the natural 5'UTR sequences of pathogen mRNAs were identified using the NCBI database. Ribosome loading and optimization design of candidate UTRs were performed using the Baidu PaddleHelix platform, and secondary structure prediction was conducted. UTRs with better prediction results were selected for validation. A DNA-RNA dual-dimensional validation system was established.

[0010] The technical solution of this invention is implemented as follows:

[0011] The present invention provides a DNA sequence that highly expresses 5'UTR-HHP1, the DNA sequence being shown in SEQ ID NO. 1.

[0012] The present invention further protects a transcribed RNA sequence of the DNA sequence that highly expresses 5'UTR-HHP1, the RNA sequence being shown in SEQ ID NO. 2.

[0013] This invention further protects the use of the above-mentioned DNA sequence that highly expresses 5'UTR-HHP1 in the preparation of drugs for treating infectious diseases, rare genetic diseases, neurodegenerative diseases, retinal diseases, cancer or tumors.

[0014] As a further improvement of the present invention, the drug is a vaccine.

[0015] This invention further protects the use of a transcribed RNA sequence of the above-mentioned DNA sequence that highly expresses 5'UTR-HHP1 in the preparation of a medicament for treating infectious diseases, rare genetic diseases, neurodegenerative diseases, retinal diseases, cancer, or tumors.

[0016] As a further improvement of the present invention, the drug is a vaccine.

[0017] The present invention has the following beneficial effects:

[0018] The 5'UTRs of genes with extremely high expression efficiency in human cells and the natural 5'UTR sequences of pathogen mRNAs were identified using the NCBI database. Ribosome loading and optimization designs were performed on candidate UTRs using the Baidu PaddleHelix platform, and secondary structure prediction was conducted. UTRs with better prediction results were selected for validation. A DNA-RNA dual-dimensional validation system was established.

[0019] At the DNA level, an enhanced green fluorescent protein (EGFP) reporter system was used to construct the pCMV-EGFP reporter plasmid, which was then amplified in TOP10 competent cells and transfected into 293T cells. The expression efficiency was evaluated using fluorescence microscopy. At the RNA level, a luciferase expression system was used to construct the pUC57 reporter vector containing the T7 promoter, 5'UTR-hbb / 5'UTR-H2O2 p1, Luciferase, 3'UTR, and polyA. After amplification with DH5α and linearization by BsaI restriction, the vector was purified by isopropanol precipitation and then transcribed in vitro using N1-methylpseudouridine triphosphate (N1-Me-pUTP) to obtain modified mRNA. Luciferase activity was detected after transfection. Because H202p1 and NS1 showed good expression, high-efficiency expression lines of hbb and H202p1 were selected and combined to construct an hbb-H202p1 combined UTR (hereinafter referred to as HHP1). After secondary structure prediction, plasmid amplification, linearization, and in vitro transcription into mRNA were performed. Luciferase activity was measured after transfection into 293T cells, showing good expression of HHP1 at the mRNA level. To further investigate the in vivo expression of HHP1, in vivo mouse imaging analysis was performed on the modified HHP1-luciferase plasmid. The constructed HHP1-luciferase mRNA was dissolved in 50 mM citrate buffer at pH 4 to achieve a final concentration of 108 ng / μL. Next, a lipid mixture was prepared: SM102, DMG-PEG2000, DSPC, and cholesterol were dissolved in anhydrous ethanol solution at a mass ratio of 50%, 1.5%, 10%, and 38.5%, respectively. The lipid-ethanol solution and the mRNA-citric acid solution were filtered separately through a 0.22 μm microporous membrane. The mRNA solution and LNP solution were mixed at a volume ratio of 3:1 to form a mixed solution. The mixture was then prepared using a microfluidic instrument. The resulting mRNA-LNP was immediately diluted with 15 mL of the above-mentioned citrate buffer and ultrafiltered using a 100 KD ultrafiltration tube at a centrifugation force of 3000 g until 1 / 4 volume was obtained. 50 mM Tris-HCl buffer (pH=7.5) was added to bring the volume to 15 mL, and this process was repeated twice. The concentration and encapsulation efficiency were then measured. Mice were vaccinated intramuscularly in the right hind limb, and in vivo imaging was performed at 24 and 48 hours post-injection. Imaging analysis showed that HHP1 was well expressed in vivo.

[0020] This invention draws on BioNTech's optimization strategy, constructing novel UTR elements by integrating the structural features of the 5' untranslated region (5'UTR) of highly expressed genes in human cells with the natural 5'UTR of pathogen mRNA. Based on deep learning methods on the Baidu PaddleHelix platform, we predicted and optimized the ribosome binding capacity of candidate UTR sequences, establishing a two-dimensional screening system: an enhanced green fluorescent protein (EGFP) reporter system at the DNA level and a luciferase expression system at the RNA level for dual verification. For UTR sequences with high expression efficiency, structural improvements were made through base modification and combinatorial optimization strategies, and microfluidic chip technology was used to precisely control the particle size distribution (80-100 nm) and encapsulation efficiency (>90%) of lipid nanoparticles (LNPs). In vitro transfection experiments showed that the optimized UTR resulted in better reporter gene expression. This research provides a new technical pathway for the subsequent engineering of UTRs in vaccines. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Predicted secondary structure diagram of hbb RNA (left); predicted secondary structure diagram of HTAT 202 p1 RNA (right);

[0023] Figure 2 This image shows the expression effect of strong green fluorescent protein (EGFP) in 293T cells after 48 hours.

[0024] Figure 3 Comparison of luciferase activity (hbb and HTAT 202 P1) 24-48 hours after mRNA transfection into HEK 293 T cells;

[0025] Figure 4 Diagram of HHP1 (hbb-HTAT202 P1 chimera);

[0026] Figure 5 Comparison of luciferase activity in HEK 293 T cells 24-48 hours after HHP1 transfection;

[0027] Figure 6 In vivo images of mice 24-48 hours after vaccination with HHP1-luciferase mRNA-LNP vaccine;

[0028] Figure 7 A comparison of bioluminescent signal intensities induced by HHP1-mRNA vaccine. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1:

[0031] PaddleHelix ribosome load prediction diagram and secondary structure (as shown in Table 1 and...) Figure 1 (As shown)

[0032] Table 1. PaddleHelix Ribosome Load Prediction Chart

[0033]

[0034] Depend on Figure 1 As shown in Table 1, after adding the Kozak sequence to hbb, the scores are close to those of other UTRs designed with PaddleHelix, so the original sequence is used. HTAT 202 p1 has the highest score, and the secondary structures of the two UTRs are significantly different.

[0035] Example 2:

[0036] DNA expression at the cellular level was validated.

[0037] At the DNA level, an enhanced green fluorescent protein (EGFP) reporter system was used to construct the pCMV-EGFP reporter plasmid. This plasmid was transfected into 293T cells using Lipofectamine 3000 transfection reagent. Expression efficiency was evaluated using fluorescence microscopy, and the results are as follows: Figure 2 .

[0038] Depend on Figure 2 It can be seen that EGFP plasmids containing UTRs produce more green fluorescence, and at the DNA level, hbb and HTAT202 P1 show better green fluorescence expression.

[0039] Example 3:

[0040] RNA cellular level validation;

[0041] At the RNA level, a luciferase expression system was used. RNA was transfected using a Mirus kit, and luciferase activity was detected after transfection. Results are as follows: Figure 3 .

[0042] Depend on Figure 3 It was found that HTAT 202 p1 showed better expression at the RNA level during cell validation. Based on the DNA-RNA cell-level results, it was confirmed that the expression of the same UTR differs between DNA and RNA; therefore, subsequent experiments will only validate at the RNA level.

[0043] Example 4:

[0044] hbb combined with HTAT 202 P1 and RNA secondary structure prediction

[0045] Based on the significant synergistic effect demonstrated by hbb and HTAT202 P1 UTR elements in previous experiments, this study employed a dual-UTR tandem strategy to construct a chimeric regulatory element. According to literature reports, the cis-arrangement of UTR elements can significantly affect their translation enhancement efficiency, with the localization strategy of highly active elements near the start codon region being more effective in improving translation efficiency. Therefore, this study used molecular cloning technology to directionally assemble hbb elements and HTAT202 P1 to construct the hbb-HTAT202 P1 chimera (hereinafter referred to as HHP1). Figure 4 ), where HTAT202 P1 is strategically positioned closer to the downstream region of the start codon to maximize its regulatory effect.

[0046] Example 5:

[0047] HHP1 RNA cellular level validation

[0048] like Figure 5 At the RNA level, a luciferase expression system was used. RNA was transfected using a Mirus kit, and luciferase activity was detected after transfection. HHP1 showed good expression performance.

[0049] Example 6:

[0050] HHP1 in vivo expression validation

[0051] At the mRNA level, HHP1 exhibited high expression efficiency. To further evaluate the application effect of the 5'UTR in the vaccine, the constructed HHP1-luciferase plasmid was linearized, and the corresponding mRNA was obtained through in vitro transcription. After preparing the vaccine using LNP-microfluidic technology, its encapsulation efficiency was measured. The vaccine was administered to mice via intramuscular injection in the right hind limb (e.g., Figure 6 In vivo imaging analysis was performed at 24 and 48 hours post-injection. Analysis results (e.g.) Figure 7 This indicates that HHP1 has a high expression level in vivo.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A DNA molecule that highly expresses 5'UTR-HHP1, characterized in that, The sequence of the DNA molecule is shown in SEQ ID NO.

1.

2. A DNA molecule that highly expresses 5'UTR-HHP1 as described in claim 1, characterized in that, The transcribed RNA sequence of the DNA molecule is shown in SEQ ID NO.

2.

3. The application of a DNA molecule that highly expresses 5'UTR-HHP1 as described in claim 1 in the preparation of an mRNA vaccine.

Images